Fig. 4

PPP1R26 is a glycolysis-associated protein and interacts with PTBP1 to control PKM2 splicing. A The correlation heatmap between expression of PPP1R26 and metabolic level in TCGA LIHC cohort. B Huh7 cells were transfected with Flag-PPP1R26 or Flag, and immunoprecipitation was performed with anti-Flag antibody on the cell extracts. PPP1R26-binding proteins were resolved by SDS-PAGE, detected by silver staining and analyzed by mass spectrometry (left). Flag or Flag-PPP1R26 was transfected into Huh7 and HepG2 cells, and immunoprecipitation was performed with anti-Flag antibody. Western blot was done with the indicated antibodies (right). C HCC cells were transfected with different dosages of Flag-PPP1R26. Western blot was done with the indicated antibodies (left). PKM2/PKM1 mRNA levels were evaluated by RT-qPCR when Flag-PPP1R26 or Flag was expressed in HepG2 and Huh7 cells (right). D PPP1R26 was depleted by shRNA and Western blot was performed to evaluate the expression of PPP1R26, PKM1 and PKM2. E PKM2/PKM1 mRNA levels were evaluated by RT-qPCR when PPP1R26 was depleted by shRNA in HepG2 and Huh7 cells (right). F Western blot analysis of indicated protein levels when GFP-PPP1R26 was expressed in the PTBP1-depleted HCC cells. G Western blot analysis of indicated protein levels in HepG2-shPPP1R26 cells or Huh7-shPPP1R26 cells transfected with GFP-PTBP1. H Huh7 and HepG2 cells were transfected with indicated PTBP1 siRNAs. Immunoprecipitation was performed with anti-PPP1R26 antibody. The immunoprecipitated RNA was submitted to RT-qPCR to evaluate the enrichment of PKM fragment flanking exon 9 by PPP1R26. The RNA enrichment was determined relative to the non-targeting IgG control. I Immunofluorescence staining was performed with anti-PPP1R26 and anti-PTBP1 antibodies. Nuclei were stained with DAPI (upper). Immunoprecipitation was performed with anti-PPP1R26 or anti-PTBP1 antibodies on Huh7 cell lysates. Rad arrows point to the heavy chain of IgG (lower). Data information: In (C, E & H), data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA with post hoc analysis LSD test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.